The first therapeutic use of corticosteroids was demonstrated in the 1950's with the introduction of cortisone acetate treatment for rheumatoid arthritis. Further studies demonstrated that the insertion of unsaturation into the 1,2 position of hydrocortisone and cortisone caused the resultant steroids, prednisolone and prednisone, to have enhanced potency and to cause less drug-induced salt retention. Subsequently, most other steroids used for the treatment of corticoid-responsive diseases have been synthesized so that they contain a double bond in the 1,2 position of the steroid molecule. In 1977, two U.S. patents were issued which represent new approaches to the synthesis of corticosteroids from sterol precursors. U.S. Pat. No. 4,035,236 covers a process for preparing 9.alpha.-hydroxyandrostenedione via fermentation of sitosterol, stigmasterol, or cholesterol. U.S. Pat. No. 4,041,055 discloses a general process for the synthesis of medically useful corticosteroids from this androstene. Intermediates covered in this chemistry can possess a 3-keto-.DELTA..sup.4,9(11) configuration.
Following are prior art methods which disclose the bioconversion of 1,2-saturated steroids to their corresponding 1,2-dehydro steroids:
(A) U.S. Pat. No. 2,837,464 "Process for Production of Dienes by Corynebacterium" PA0 (B) U.S. Pat. No 3,360,439 entitled "Process for Preparing 1-dehydro Steroids". PA0 (C) Charney, W. and Herzog, H. 1967. Microbial Transformation of Steroids. Academic Press, Inc., New York, pp 4-9, 236-261. PA0 (D) T. Yamane, H. Nakatani, E. Sada, T. Omata, A. Tanaka and S. Fukui, Biotechnology and Bioengineering, 21. 1979. PA0 (E) K. Sonomoto, I. Jin, A. Tanaka and S. Fukui; Agric. Biol. Chem., 44, 1119-1126, 1980.
Description of 1-dehydrogenation of steroids in fermentation beers by Arthrobacter (Corynebacterium) simplex.
Description of 1-dehydrogenation of steroids by use of A. simplex cells pretreated with a lower alkanol or lower alkanone such as acetone before mixing with the substrate and a hydrogen carrier.
Historical background on steroid bioconversions and taxonomic listing of microorganisms known to carry out 1-dehydrogenation.
The authors studied the .DELTA..sup.1 -dehydrogenation of 4-androstene-3,17 dione with Nocardia rhodocrous.
They used 50% benzene-heptane as a solvent. They report that the advantage of using the solvent is that it increases the reaction rate by a factor of 180. (Benzene is a human carcinogen).
It should be noted that this enzyme must be different than that produced by A. simplex. The Nocardia rhodocrous enzyme is not active in the presence of menadione, the electron acceptor of choice for A. simplex.
The preferred electron acceptor for the Nocardi rhodocrous is phenazine methosulfate. This electron acceptor is too expensive and unstable to render this microbial activity practical.
They have also run the reaction with the solvent only 20% saturated with the steroid. They demonstrate that increasing the concentration of the steroid decreases the reaction rate.
The solvent 50% benzene-heptane is flammable in the presence of oxygen. Oxygen is an essential reagent of the reaction. The authors did not discuss how oxygen could be introduced without producing an explosive environment in the reactor.
The same lab studied the .DELTA..sup.1 -dehydrogenation of hydrocortisone with A. simplex. They considered two immiscible solvents, n-butanol and n-amyl alcohol, and reported that the reaction did not proceed in the presence of these solvents. They preferred water miscible solvents and chose 10% methanol-water and 10% propylene glycol-water as their solvents.